The invention relates to the field of vascular regulation and to the role of R-Ras in vascular regulation. The invention in particular relates to means and methods for promoting a quiescent state for a vascular cell and to means and methods for diagnosing a condition of vasculature of an individual. More in particular the invention relates to RAS proteins or functional equivalents thereof, as well as their genes, particularly in gene therapy settings for providing RAS-activity to vascular cells.
The Ras family of small GTPases comprises intracellular signaling molecules that function as binary switches regulated by GTP. The oncogenic Ras proteins, H-Ras, and K-Ras in particular, are central players in cellular signaling networks; they are activated by most growth factors as well as by integrins. R-Ras is a member of the Ras protein family that antagonizes H-Ras signaling1,2. Due to its sequence homology with the prototypic Ras proteins, R-Ras has been often described as a transforming oncogene3. The transforming activity of R-Ras in vitro is, however, quite low compared with that of H-Ras and K-Ras4, and there is no evidence that activating mutations would occur in spontaneous malignancies. Moreover, R-Ras differs from the other members of the Ras family in that it contains a proline-rich SH3 domain binding site, within which it can be phosphorylated by Eph receptors and Src; both SH3 domain binding and phosphorylation regulate R-Ras activity5-7.
The present invention makes use of the finding that R-Ras has different properties than the more conventional Ras proteins/genes. R-Ras and H-Ras for instance exert opposite effects on cell-extracellular matrix adhesion; R-Ras enhances integrin-mediated cell adhesion by elevating the affinity and avidity of integrins8, whereas H-Ras inhibits integrin activities9. There is also a striking contrast in R-Ras and H-Ras activities in cell differentiation. R-Ras promotes the differentiation of myoblasts and the fusion of these cells to myotubes, a process that requires cell cycle arrest and entry to G0 state10, whereas H-Ras inhibits these processes11. The in vivo function of R-Ras, however, has formerly remained unclear. The invention surprisingly discloses that R-Ras is a vascular regulator. In particular, R-Ras is a regulator of vascular differentiation that primarily affects the remodeling of blood vessels.
The invention in one embodiment provides a method for promoting a quiescent state for a vascular cell, comprising providing said cell with additional R-Ras activity. A quiescent state for a vascular cell as used in the invention is a state wherein the cell cycle of a vascular cell is arrested and/or wherein a cell is in the GO state. Promoting means that the process of cell cycle arrest is enhanced and/or that the entrance of a cell into the GO state is stimulated. Further, promoting means that a cell that is in a quiescent state is stimulated to maintain the quiescent state. A quiescent state of a vascular cell means that cell proliferation and migration is inhibited and further that the cell is in a state that facilitates differentiation. There are many instances wherein it would be very favourable to promote a quiescent cell state in a vascular cell. For instance, in tumors, in hyperplastic arterial lesions, in pulmonary hypertension, pulmonary vascular diseases, organ transplant rejection, peripheral artery disease, coronary artery disease, collagen vascular diseases such as lupus and scleroderma, vasculitis and autoimmune diseases. An additional R-Ras activity is any amount of activity that raises the activity of a cell above the initial level. Thus, in a cell wherein R-Ras activity was for example absent, or alternatively, was present but at a lower level. A vascular cell as used in the invention is preferably an endothelial cell or a smooth muscle cell. R-Ras activity can be added by any means or method that enhances the presence or activity of R-Ras or a functional equivalent thereof in a vascular cell. This can be achieved directly by providing R-Ras protein or a functional equivalent thereof or indirectly by providing an expressible nucleic acid which can express R-Ras activity and/or by activating a pathway in a vascular cell that leads to expression of R-Ras activity (in particular of a R-Ras protein), or activation of pre-existing R-Ras.
In one embodiment of the invention additional R-Ras activity is provided through activation of inactive R-Ras in a cell. The activity of Ras proteins is in one aspect controlled by the balance of GTP and GDP bound to the protein, wherein the GTP-bound form is active and the GDP-bound form is inactive. Activation is for instance established by stimulating the binding of GTP to the R-Ras protein. In one embodiment of the invention additional R-Ras activity is provided by converting GDP-bound R-Ras protein into GTP-bound R-Ras protein, by phosphorylating said GDP. As the R-Ras protein itself is a GTPase, its activity is further regulated by accessory proteins that influence the activity of the GTPase functionality. In a further embodiment of the invention additional R-Ras activity is thus provided by inhibiting or enhancing presence and/or activity of accessory proteins. In an alternative embodiment of the invention post-translational modification by lipid attachment is enhanced to provide additional R-Ras activity or post-translational modification by lipid attachment is inhibited to reduce R-Ras activity. Post-translational modification of R-Ras protein in order to make the protein functional is, for instance, farnesyl or geranyl attachment. R-Ras has a further regulation mechanism that is typical for R-Ras compared to the other Ras proteins. R-Ras differs from the other members of the Ras family in that it contains a proline-rich SH3 domain binding site, within which it can be phosphorylated by Eph receptors and Src. Eph receptors and Src are both protein tyrosine kinases (PTKs). Protein tyrosine kinases (PTKs) are enzymes which catalyze the phosphorylation of tyrosine residues. PTKs are involved in cellular signaling pathways and regulate key cell functions such as proliferation, differentiation, anti-apoptotic signaling and neurite outgrowth. There are two main classes of PTKs: receptor PTKs and cellular, or non-receptor, PTKs. Cellular PTKs, such as Src, are located in the cytoplasm, nucleus or anchored to the inner leaflet of the plasma membrane. Receptor PTKs possess an extracellular ligand binding domain, a transmembrane domain and an intracellular catalytic domain. Eph receptors form the largest subfamily of receptor tyrosine kinases (RTKs). Both SH3 domain binding and phosphorylation regulate R-Ras activity5-7. Thus in one embodiment of the invention R-Ras activity is added by enhancing binding of SH3 domains to a proline-rich site of R-Ras. In a preferred embodiment, regulation of R-Ras activity is provided by enhancing or inhibiting presence of R-Ras protein, as the amount of protein is a stronger regulator of R-Ras activity in a cell than internal activating or inhibiting mechanisms of R-Ras protein.
The invention in a preferred embodiment provides a method according to the invention, wherein said activity is provided by an R-RAS protein and/or a functional equivalent thereof. A functional equivalent is any equivalent having the same function in kind, not necessarily in amount. A sequence of wild type R-Ras protein is given in FIG. 14. For further characterization and expression of the Human R-Ras gene product, see for example ref. 51. R-Ras proteins have little or no species specificity. Wild type and mutant R-Ras proteins found in an animal are thus applicable in many other species. A functional equivalent is for example a synthesized artificial R-Ras protein, a recombinant and/or a mutant R-Ras protein. Examples of such mutants are R-Ras 38V and R-Ras 87L. Such mutants are optionally combined to derive a potent R-Ras protein. In another embodiment of the invention additional R-Ras activity is provided by introduction of a nucleic acid encoding an R-Ras protein or a functional equivalent thereof. An alternative means to provide additional R-Ras activity is enhancing R-Ras gene transcription and/or translation e.g. by stimulating a promoter sequence of R-Ras.
In an alternative embodiment of the invention R-Ras activity in a vascular cell is reduced. Reduction of R-Ras activity in a vascular cell is for example established by down-regulating R-Ras expression in the cell or by degrading R-Ras protein and/or R-Ras mRNA in a cell, and by promoting hydrolysis of GTP bound to R-Ras to GDP or by preventing the modification of R-Ras by lipid attachment. Down-regulating is for example established by an antisense-technique. Non-limiting examples of some of the newer antisense approaches are interference RNA (RNAi), microRNA and splice interference techniques such as exon-skipping. Reducing R-Ras activity in a cell stimulates proliferation and migration and diminishes differentiation of the cell. Angiogenesis is thus stimulated by down-regulation of R-Ras activity. Indications for down-regulating R-Ras activity in a cell are for example rehabilitation of an infarct, a stroke or of organ or limb damage. Balancing down- and up-regulation of R-Ras activity is a method for structuring modeling of vasculature. The invention in one embodiment provides a method for structuring modeling of vasculature, comprising providing said cell with additional R-Ras activity or reducing R-Ras activity in said cell. Modeling herein proceeds through stimulation/inhibition of cell growth, differentiation, proliferation and migration.
In one embodiment of the invention, R-Ras activity is added or reduced by intervening in an R-Ras pathway. The results of examples of the invention demonstrate that the effect of R-Ras on vascular cell activities is mediated, at least in part, by inhibitory phosphorylation of Raf-1. Other pathways do, however, also play a role. In a preferred embodiment of the invention R-Ras activity is added by enhancing inhibitory phosphorylation of Raf-1, or reduced by diminishing inhibitory phosphorylation of Raf-1. Raf-1 is at least part of a PI3-kinase-Akt signaling pathway. In one embodiment of the invention R-Ras activity is enhanced by activating at least a subset of Akt molecules or reduced by inhibiting at least a subset of Akt molecules.
An R-Ras protein is produced in any prokaryotic or eukaryotic cell. Delivering an R-Ras protein to a cell is done by any means that transports the protein/peptide into a cell. For example by electrotransfection, electroporation or through delivery by a cell penetrating peptide. Alternatively, R-Ras protein is delivered comprised in a nanoparticle. In a preferred embodiment the invention provides a method according to the invention, wherein said R-Ras protein or said functional equivalent thereof is provided with a cell penetrating peptide. There are many types of cell penetrating peptides available in the art. For references, see for example ref. 52-55. Any type of cell penetrating peptide that delivers R-Ras protein or a functional equivalent thereof can be used according to the invention. A cell penetrating peptide is for example a protein derived peptide, such as penetratin, a Tat-derived peptide, a signal-sequence-based peptide (I or II), or a synthetic and/or chimeric cell-penetrating peptide such as transportan or an amphiphilic model peptide. Specific preferred examples of such cell penetrating peptides are Tat, a cell penetrating peptide (refs. 96-98); F3 (ref. 95), an internalizing tumor specific homing peptide; and Arginine Heptamer (refs. 99-104), which is a dermis penetrating peptide.
In a further preferred embodiment the invention provides a method according to the invention, wherein said R-Ras protein or said functional equivalent is provided with a homing peptide. A homing peptide is any peptide that targets a cell of a selected tissue. In the art many homing peptides are available. In a preferred embodiment the homing peptides are lung homing peptides, heart homing peptides or tumor homing peptides. Heart homing peptides are for example a CRPPR (SEQ ID NO:1) peptide that at least binds to a Cysteine-rich protein 2 receptor, and a CPKTRRVPC (SEQ ID NO:2) peptide that at least binds to a bc10 receptor. For further references for heart homing peptides see for instance ref 56, specifically the table on page 1605. Lung homing peptides are for example Metadherin or GFE-1 (CGFECVRQCPERC (SEQ ID NO:3)). For further references for lung homing peptides see for instance refs 57, 58 and 105-110. Preferred tumor-homing peptides are for example F3, a 34-amino acid basic peptide (see further refs. 59, 60); CGKRK (SEQ ID NO:4) (see under more refs. 61 and 62); and LyP-1, sequence CGNKRTRGC (SEQ ID NO:5) (see for instance refs. 63 and 64). Relevant tumor homing peptides are further for instance described in refs 59-64. Preferably a homing peptide homes to lung, heart, especially a coronary artery, or any vasculature. The cell penetrating peptides and/or homing peptides can be attached to the protein providing R-Ras activity by any conventional means. An easy way of preparing the complex is through producing a vector encoding a fusion of R-Ras protein with a cell penetrating peptide and/or a homing peptide and expressing said vector in a suitable host as disclosed in the examples.
In one embodiment the invention provides R-Ras or a functional equivalent thereof for use as a medicine. The medicine is used for any occasion wherein it is desired to promote a quiescent state for a vascular cell. In a preferred embodiment R-Ras or a functional equivalent thereof is used in the preparation of a medicament to promote a quiescent state for a vascular cell. A medicament is any pharmaceutical composition comprising R-Ras or a functional equivalent thereof. Such a pharmaceutical composition optionally further comprises a pharmaceutically acceptable carrier and any additive. A medicine is for example produced as a pill, a capsule, a tablet, a medicinal gum, a solution, a dry powder form, an inhaler, transdermal patch, microsphere, nanocrystal, or as a coating for a device that is introduced in a body within contact of vasculature.
In one embodiment the invention provides a protein complex comprising R-Ras or a proteinaceous functional equivalent thereof and a cell penetrating peptide and/or a homing peptide. Such a protein complex is for example a fusion protein or a complex which is held together by disulfide bonds. In a preferred aspect of the invention a protein complex according to the invention is a fusion protein. There are diverse chemical coupling methods available in the art for the production of a fusion protein, a fusion protein of the invention is produced by any of these methods. An example of such a fusion protein is a fusion protein of R-Ras or R-Ras mutant 38V or 87L and a Tat-derived peptide. In one embodiment of the invention, a homing peptide is a part of a fusion protein. A functional equivalent as used in the invention is at least functionally the same in kind as R-Ras, though not necessarily the same in amount. The invention further provides a nucleic acid molecule comprising a nucleic acid sequence coding for a fusion protein according to the invention. A nucleic acid is for example a DNA, RNA or PNA. In an alternative embodiment of the invention, R-Ras is comprised in a nanoparticle. For this embodiment, R-Ras does not have to be modified, but can be if desired. A homing peptide is optionally attached to the particle core.
In one aspect the invention provides a gene delivery vehicle encoding R-Ras or a functional equivalent thereof. In a further embodiment the invention provides a gene delivery vehicle encoding a fusion protein according to the invention, or comprising a nucleic acid molecule according to the invention. A gene delivery vehicle is any vehicle that delivers a gene encoding an R-Ras or functional equivalent thereof to a cell. Preferably said cell is a vascular cell, more preferably an endothelial or a smooth muscle cell. A gene delivery vehicle is in a preferred embodiment a vector encoding R-Ras or a functional equivalent thereof. A vector is preferably an expression vector wherein R-Ras or a functional equivalent thereof is operatively linked to an enhancer sequence and/or a promoter sequence. Transduction of a vector according to the invention is for example mediated through a virus. Non-limiting examples of such viruses are an adenovirus, an adeno associated virus, an alphavirus such as sindbis or SFV a lentivirus and/or a retrovirus. Non-viral delivery vehicles include polymers and/or liposomes or other nanoparticles (based on e.g. biodegradable polymers).
In a preferred embodiment the invention provides an article of manufacture to be introduced into an environment comprising vascular cells, coated with a composition comprising R-Ras or a functional equivalent thereof, or a composition comprising a protein complex according to the invention, or a composition comprising a gene delivery vehicle according to the invention. Coated as used in the invention means that the additional R-Ras activity is available to the environment comprising vascular cells. Coated means preferably, but not necessarily, that the layer of the article of manufacture exposed to the environment is provided with additional R-Ras activity. Such an article of manufacture is introduced in any environment wherein additional R-Ras activity is beneficial. Additional R-Ras activity is for example beneficial in an environment where cell migration and proliferation, in particular of vascular cells, is undesirable. Providing additional R-Ras activity is for example beneficial in order to prevent a hyper-reactive response of an organism to an external device that is inserted in said organism. Adding R-Ras activity at least partially inhibits vascular cell proliferation and migration as a response to an external stimulus. Another example of a circumstance wherein adding R-Ras activity is beneficial, is in the case of tumor growth. R-Ras activity then prevents angiogenesis in and adjacent to the tumor, thereby inhibiting the blood supply to the tumor tissue. In addition, adding R-Ras activity increases cell adhesion. In one embodiment the invention provides use of R-Ras or a functional equivalent thereof, for stimulating adhesion of vascular endothelial cells to a vessel wall. Increasing cell adhesion can further, for instance, convert a leukemia into a lymphoma or inhibit metastasis. Increasing cell adhesion through R-Ras is at least partially mediated by integrins. In a preferred embodiment the invention provides an article of manufacture to be introduced into an environment comprising vascular cells, coated with a composition comprising R-Ras or a functional equivalent thereof, or a composition comprising a protein complex according to the invention, or a composition comprising a gene delivery vehicle according to the invention, further comprising a Tat peptide and/or a homing peptide. In a further preferred embodiment said article of manufacture is a stent.
An article of manufacture of the invention is any article of manufacture that is in any instance introduced into an environment comprising vascular cells. Such an article is for example a device that forms a portal in a mammal. Such a portal is for instance a portal to a bowel, a stomach, a bladder, an esophagus or a trachea. Alternatively, an article of the invention is a device that is placed internally in an environment that comprises vascular cells. An internally placed article is for example an artificial heart-valve. In a preferred embodiment of the invention an article of manufacture according to the invention is a stent. A stent as used in the invention is a device that is open in that there is passage possible for bodily fluids and that is inserted in an organism, preferably a mammal. A stent is preferably a small tube that is inserted for more than 6 hours, preferably for longer, in a preferred embodiment permanently, in an organism. In a preferred embodiment the stent is introduced in an artery. The stent at least assists in holding open an artery so that blood can flow through it. In a preferred aspect a stent is an intraluminal coronary artery stent. Such a stent is typically used in angioplasty, which is a procedure to reduce or eliminate blockages in coronary arteries.
In one embodiment the invention provides a composition for promoting a quiescent state for a vascular cell, comprising R-Ras or a functional equivalent thereof, a protein complex according to the invention, or a gene delivery vehicle according to the invention.
Such compositions can be pharmaceutical compositions, cosmetic compositions or compositions for use on cells in vitro. These compositions can therefore comprise the conventional usual excipients, buffers and other constituents for such compositions. The invention in another embodiment provides a composition according to the invention, further comprising a 2nd/3rd active agent. In a further embodiment the invention provides such a composition whereto a 4th, 5th, or any further active agent is added. An active agent is any active agent that is beneficially combined with R-Ras activity in a composition. Such a composition is for instance a pharmaceutical that is intended for treatment and/or prevention of vascular narrowing. Further active agents in the composition are, for that indication, for instance heparin or another anticoagulant or acetylsalicylic acid. Vascular narrowing is for example a problem in a patient with post-angioplasty restenosis or artherosclerosis. In a further preferred embodiment the invention provides a composition according to the invention, further comprising sildenafil citrate, sirolimus, and/or Gleevec.
Preferred active agents in a composition of the invention are:                Diuretics, sometimes called “water pills”. These agents flush excess water and sodium from the body by increasing urination. This reduces the amount of fluid in the blood and flushes sodium from the blood vessels so that they can open wider, increasing blood flow and thus reducing the blood's pressure against the vessels. Diuretics are optionally used in combination with other high blood pressure drugs. Types of diuretics include thiazides, such as Diuril (chlorothiazide) and Esidrex (hydrochlorothiazide); potassium-sparing diuretics, such as Aldactone (spironolactone); and loop diuretics, such as Lasix (furosemide).        Beta blockers. These agents slow the heartbeat by blocking the effect of nerve impulses to the heart and blood vessels, thereby lessening the burden on the heart. Beta blockers include Inderal (propranolol), Lopressor (metoprolol), and Tenormin (atenolol).        ACE (angiotensin-converting enzyme) inhibitors. These agents inhibit formation of the hormone angiotensin II, which causes blood vessels to narrow, thus increasing blood pressure. ACE inhibitors include Altace (ramipril), Capoten (captopril), and Zestril (lisinopril).        Calcium channel blockers. These agents prevent calcium from entering the muscle cells of the heart and blood vessels, thus relaxing blood vessels and decreasing blood pressure. Some calcium channel blockers are Procardia (nifedipine), Isoptin (verapamil) and Cardizem (diltiazem).        Alpha-beta blockers. These agents combine the actions of alpha blockers, which relax blood vessels, and beta blockers, which slow the heartbeat. The dual effect reduces the amount—and thus pressure—of blood through blood vessels. Alpha-beta blockers include Normodyne and Trandate (both labetalol).Diuretics, beta blockers, ACE inhibitors, calcium channel blockers and alpha-beta blockers and functional analogues and derivatives of these agents are preferably used in the case of hypertension, more preferably in the case of systemic hypertension. The functional analogues or derivatives as used in the invention have a function that is the same in kind, not necessarily the same in amount.        
Active agents that are specifically preferred in a composition of the invention for use in case of pulmonary hypertension are: epoprostenol, treprostinil, bosentan, sildenafil, iloprost, sitaxsentan, and Ambrisentan, or functional analogues or derivatives thereof. Preferred active agents in a composition of the invention for use in case of need of an anti-proliferative, for instance in cancer, are: Methotrexate, Fluorouracil, cisplatin, doxorubicin, 5-fluotacil; VEGF inhibitors such as bevacizumab (Avastin); Protein kinase inhibitors such as imatinib (Gleevec); and epidermal growth factor receptor tyrosine kinase inhibitors such as erlotinib (Tarceva) and gefitinib (Iressa), or functional analogues or derivatives of these agents. Preferred agents in a composition of the invention for use in stent coatings are: Sirolimus, Tacrolimus, Paclitaxel, Everolimus, or functional analogues or derivatives thereof.
In one embodiment a composition according to the invention is a pharmaceutical composition optionally further comprising additives, such as a pharmaceutically acceptable carrier or a colorant.
In a preferred embodiment the invention provides a composition, which is a controlled release composition. A controlled release composition as used in the invention is any composition that does not release all activity at one point in time. A controlled release composition of the invention preferably constantly releases approximately even amounts of active agent for a longer period of time. A longer period of time are preferably hours, more preferably days, and most preferred weeks, months or years. A controlled release composition of the invention is typically a biodegradable composition. A controlled release composition is for example produced in the form of a biodegradable microsphere (see for example ref. 65). A controlled release composition of the invention is for example used in a coated article of manufacture of the invention, such as a stent. Adding a controlled release composition of the invention to for instance a heart atrioventricular valve will reduce vessel proliferation on implantation of the valve in for example a mammal. Thereby adding such a composition at least partially helps to prevent the forming of an obstructive mass in a blood stream.
In one embodiment the invention provides a method for diagnosing a condition of vasculature of an individual, comprising obtaining a sample from said individual and measuring an expression level of R-Ras expression in said sample. A condition of vasculature as used herein is a status of health or development of vasculature of an individual, such as a pathological or a physiological status. Furthermore the status refers characteristics of the vasculature such as the differentiation state of a cell or of multiple cells. A sample as used in the invention is for example a sample of a bodily fluid, such as blood or lymph. A sample is alternatively obtained from a bronchoalveolar lavage (BAL), Transbronchial biopsy (TBB), or Endomyocardial heart biopsy. In a preferred embodiment a sample is a vascular cellular sample. A vascular cellular sample of the invention is any sample comprising cells that were located adjacent to or part of a vascular tissue. A vascular cellular sample is preferably derived from one of the larger vessels in an animal. Preferably said larger vessel is an artery. In a preferred embodiment a vascular cellular sample is obtained from a mammal, preferably a human being. A vascular sample as used in the invention is obtained with any method for taking a vascular cellular sample. Such a method is for example a surgical method or a minimal invasive method such as a cheek mucosal tissue sampling technique. In a preferred embodiment of the invention a sample in a method for diagnosing a condition of vasculature of an individual is obtained with a percutaneous endoarterial biopsy, a percutaneous atherectomy, or another surgical method.
An expression level of R-Ras can be measured in alternative ways. An expression level of R-Ras can be measured from any product of an R-Ras mRNA. For example the level of R-Ras protein, or the level of a derivative of R-Ras protein is measured. An expression level of R-Ras is for example performed through an immunodetection technique, such as immunohistochemistry, immunofluorescence or immunoblotting. Alternatively expression levels are determined with a PCR technique, for instance quantitative real time PCR. In the art many other techniques for determining an expression level are available, such as multiple microarray techniques. In a preferred embodiment a method according to the invention is provided, wherein measuring is performed through PCR, a microarray technique, immunohistochemistry, immunofluorescence or immunoblotting.
In one embodiment the invention provides a method for diagnosing a condition of vasculature of an individual, wherein said condition of vasculature of said individual is associated with a disorder in said individual and wherein said disorder is a vascular proliferative disease. Diagnosis is either directed to a local, a regional or a systemic condition of vasculature of an individual. A vascular proliferative disorder is any disease wherein vasculature of an individual proliferates. Proliferation typically refers to cell multiplication, but generally, as in most vascular proliferative disorders, it also involves growth of at least some individual cells. Non-limiting examples of vascular proliferative disorders are: idiopathic pulmonary hypertension, chronic hypoxic pulmonary hypertension, systemic hypertension, atherosclerosis, post-angioplasty restenosis, vasculopathy, diabetic vasculopathy, vascular injury, vasculitis, arteritis, capillaritis or carcinoma. In a preferred embodiment, the invention provides a method for diagnosing a condition of vasculature of an individual, wherein said vascular proliferative disease is selected from the following: pulmonary hypertension, carcinoma or vascular injury.
The invention provides a kit for diagnosing a condition of vasculature of an individual, at least comprising a means for measuring an expression level of R-Ras in a sample and an apparatus for obtaining a sample. An apparatus for obtaining a sample is any device that can be used to derive a sample as defined by the invention. A non-limiting example of such an apparatus is a syringe or syringe-like device or a catheter or biopsy device. In a preferred embodiment of the invention, an apparatus for obtaining a sample is an atherectomy catheter or an endoarterial biopsy device.
The invention further provides a kit according to the invention, wherein the means for measuring an expression level comprises a binding body and a detection reagent. Many different specific binding bodies are available. Of old, antibodies are used. However, currently many different parts, derivatives and/or analogues of antibodies are in use. Non-limiting examples of such parts, derivatives and/or analogues are, single chain Fv-fragments, monobodies, VHH, Fab-fragments and the like. A common denominator of such specific binding bodies is the presence of an affinity region (a binding peptide) that is present on a structural body that provides the correct structure for presenting the binding peptide. Binding peptides are typically derived from or similar to CDR sequences (typically CDR3 sequences) of antibodies, whereas the structure providing body is typically derived from or similar to framework regions of antibodies. A detection reagent is any reagent that provides detection of binding or non-binding of an antibody. A detection reagent for example comprises a reporter group or is a fluorescent marker.